Colored microsphere probe as well as preparation method and application thereof

Abstract

The invention provides a colored microsphere probe as well as a preparation method and application thereof, and belongs to the technical field of biological detection. The invention provides a preparation method of a colored microsphere probe, which comprises the following steps: cleaning colored microspheres, and centrifuging to obtain a precipitate; activating the microspheres in the precipitate; re-suspending the microspheres by using a boric acid buffer solution after cleaning again; coupling an osteocalcin antibody on the resuspended microspheres, and then sealing and storing. According to the invention, abundant carboxyl groups on the surface of the colored microspheres are coupled with a specific antibody to form a stable immune complex which is used as a tracing marker for detecting osteocalcin, so that a powerful tool is provided for diagnosis of clinical bone metabolism related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a colored microsphere probe and a preparation method and application thereof. Background Art

[0002] Osteocalcin is a non-collagenous bone matrix protein specifically synthesized and secreted by non-proliferative osteoblasts in bones, and osteocalcin plays a key role in the bone mineralization process. However, the existing osteocalcin detection methods have high detection costs and require large instruments. They are not suitable for bedside detection, remote areas, and home use, which affects the preliminary screening of bone metabolism-related diseases. In order to solve the above problems, the present invention proposes a quantitative detection method and kit for osteocalcin based on carboxyl colored microspheres. The quantitative detection method and kit for osteocalcin based on carboxyl colored microspheres of the present invention have important clinical application value and scientific research significance, and provide reliable technical support for the early diagnosis and treatment of bone metabolism-related diseases. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a colored microsphere probe, and a test strip prepared by the method can quantitatively detect osteocalcin to meet the high requirements of clinical and scientific research for osteocalcin detection.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a colored microsphere probe, comprising the following steps:

[0006] (1) washing the colored microspheres and centrifuging to obtain a precipitate;

[0007] (2) Activating the microspheres in the precipitate;

[0008] (3) After washing again, resuspend the microspheres in borate buffer;

[0009] (4) The resuspended microspheres are coupled with osteocalcin antibodies, and then blocked and stored.

[0010] Preferably, the cleaning in step (1) is performed using a boric acid buffer solution at pH 7.4-pH 8.0, and the volume ratio of the colored microspheres to the boric acid buffer solution is 1-10:100.

[0011] Preferably, the activation in step (2) is to resuspend the precipitated microspheres in a boric acid buffer at pH 7.4-pH 8.0, add EDC solution and NHS solution, and continue in a metal bath at 35° C.-39° C. for 25-35 minutes.

[0012] Preferably, during the activation, the concentration of the EDC solution is 0.8-1.2 mg / ml, the concentration of the NHS solution is 0.8-1.2 mg / ml, and the volume ratio of the boric acid buffer, the EDC solution and the NHS solution is 90-110:2.2-2.6:2.6-3.

[0013] Preferably, the second washing in step (3) is performed using a boric acid buffer having a pH value of 7.4 to 8.0, wherein the volume of the boric acid buffer added is 4 to 6 times the volume of the boric acid buffer added during activation. After the washing, the mixture is centrifuged again and resuspended with a boric acid buffer having a pH value of 7.4 to 8.0, wherein the volume of the resuspending buffer is 1 / 9 to 1 / 10 of the volume of the boric acid buffer used during the above washing.

[0014] Preferably, the antibody in step (4) is K131c1, the concentration of K131c1 is 3.5-4.5 mg / mL, the volume ratio of K131c1 added to the resuspension buffer is 1-3:50-60, the coupling conditions are in a metal bath at 35°C-39°C for 55-65 min, the blocking is followed by centrifugation, and then the preservation solution is added, the preservation solution is a boric acid buffer solution of 1-3% BSA pH 7.4-pH 8.0.

[0015] The invention also provides a colored microsphere probe obtained by the preparation method.

[0016] The present invention also provides a test strip prepared by the colored microsphere probe.

[0017] Preferably, the test strip comprises a nitrocellulose membrane, a sample pad and absorbent paper attached to a bottom plate, and the detection line of the nitrocellulose membrane is coupled with the osteocalcin antibody K135c2.

[0018] The present invention utilizes the abundant carboxyl groups on the surface of colored microspheres to couple with specific antibodies to form a stable immune complex as a tracer marker for detecting osteocalcin, providing a powerful tool for the diagnosis of clinical bone metabolism-related diseases. Compared with colloidal gold technology, the osteocalcin immunochromatographic test strip of colored microspheres provided by the present invention has higher sensitivity and can meet the high requirements of clinical and scientific research for osteocalcin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the osteocalcin test strip with colored microspheres in Example 2;

[0020] Figure 2 The results of transmission electron microscopy identification of osteocalcin-coupled colored microspheres in Example 3; A. Transmission electron microscopy image of colored microspheres not coupled with osteocalcin antibody; B. Transmission electron microscopy image of colored microspheres-osteocalcin antibody coupled;

[0021] Figure 3The zeta potential identification result of osteocalcin-coupled colored microspheres in Example 4;

[0022] Figure 4 The specific detection result of the osteocalcin colored microsphere test strip in Example 5;

[0023] Figure 5 The results of the osteocalcin colloidal gold immunochromatographic test strip test in Comparative Example 1 are as follows;

[0024] Figure 6 The detection range of the osteocalcin immunochromatographic test strip of the colored microspheres in Comparative Example 1;

[0025] Figure 7 This is a comparison of the results of three colored microsphere probes in comparative example 2; A. Comparison of the results of preparing colored microsphere probes using three coupling buffers; B. Comparison of the effects of three colored microsphere probe test strips. DETAILED DESCRIPTION

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Example 1

[0028] Preparation of colored microsphere probes

[0029] A. Cleaning:

[0030] (1) Pipette 5 μL of red colored microspheres (DR0300CA, Suzhou Weidu Biotechnology Co., Ltd.) and add them into a 1.5 mL conical bottom centrifuge tube.

[0031] (2) Add 100 μL of pH 7.4 boric acid (BB) buffer (0.05 M borax (1 mL) + 0.2 M boric acid (9 mL)), mix by pipetting for 15 s, centrifuge at 20,000 g / min for 5 min, remove the supernatant, and obtain the precipitated microspheres.

[0032] B. Activation:

[0033] (1) Resuspend the washed microspheres in 100 μL of pH 7.4 BB buffer and mix by pipetting.

[0034] (2) Take 2.4 μL of EDC solution (1 mg / ml) and add it to the above solution. Then add 2.8 μL of NHS (1 mg / ml), mix by pipetting, and place in a metal bath at 37°C for 30 min, shaking every 10 min.

[0035] C. Cleaning:

[0036] (1) Add 500 μL of pH 7.4 BB buffer, mix by pipetting, centrifuge at 20,000 g / min for 15 min, and remove the supernatant;

[0037] (2) Add 50 μL of pH 7.4 BB buffer to resuspend the microspheres.

[0038] D. Conjugated osteocalcin antibody:

[0039] Add 3 μL of K131c1 antibody (4 mg / mL, Nanjing Oukai Biotechnology Co., Ltd.) to the resuspended microsphere solution, mix well by pipetting, and then place in a 37°C metal bath for 1 h.

[0040] E. Closure:

[0041] Add 10 μL of blocking solution (10% BSA) and react at room temperature (20-25°C) for 30 min, shaking every 10 min.

[0042] F. Save:

[0043] The blocked solution was centrifuged at 20000g / min for 15min, and the supernatant was discarded. 100μL of preservation solution (BB buffer containing 1% BSA pH 7.4) was added, and stored at 4°C in the dark. When used, it was diluted 10 times with carbonate buffer containing 1% Tween 20 pH 9.6 (CBS, Beijing Solebow Technology Co., Ltd., 50mM).

[0044] Example 2

[0045] Preparation of osteocalcin test strips

[0046] (1) Glue the nitrocellulose membrane (NC membrane), sample pad, and absorbent paper to the base plate in sequence, with the NC membrane located in the middle of the base plate, and the sample pad and absorbent pad located at the left and right ends of the NC membrane and covering it by about 3 mm for lamination. Use a paper cutter to cut it into strips of approximately 4 mm each. Place the prepared test strips in a sealed bag and store them at room temperature away from light.

[0047] (2) Osteocalcin antibody (K135c2) was used as the coating source for the detection line (T line), and goat anti-mouse IgG was used as the coating source for the quality control line (C line), and the samples were spotted on the NC membrane and incubated in an incubator to fix the antibodies on the T line and C line to obtain a colored microsphere osteocalcin test strip ( Figure 1 ).

[0048] The principle of detecting osteocalcin by colored microspheres: Place the sample, colored microsphere probe and loading buffer in the microwell of the ELISA plate, mix and let it stand, then insert the test strip into the microwell (the sample pad contacts the liquid first). Using capillary action, the sample will flow on the test strip for chromatography. When the liquid flows through the T line, if the sample contains osteocalcin, it will bind to the osteocalcin antibody (K131c1) on the colored microsphere probe, and then bind to the osteocalcin antibody (K135c2) on the T line to form a double antibody sandwich antigen complex. The excess colored microsphere probe continues to flow to one end of the absorbent paper of the test strip, flows through the C line, and binds to the sheep anti-mouse IgG antibody on the C line to develop color. On the contrary, if the sample does not contain osteocalcin, the T line will not develop color, and only the C line will develop color. After the chromatography is completed, take out the test strip from the enzyme-labeled well, observe the results with the naked eye, and record the results by taking a photo with a mobile phone. Within a certain range, the higher the content of osteocalcin in the sample, the darker the color of the T line.

[0049] Example 3

[0050] Transmission electron microscopy identification of osteocalcin-coupled colored microspheres

[0051] (1) Take 10 μL of bare colored microspheres and 10 μL of the colored microsphere probe prepared in Example 1.

[0052] (2) Add the above solutions dropwise onto a copper mesh (200 mesh carbon-free square film) and let stand for 10 min.

[0053] (3) Use the edge of the filter paper strip to absorb excess sample liquid from the edge of the copper mesh and dry at room temperature until there is no liquid on the surface of the copper mesh.

[0054] (4) Drop 10 μL of phosphotungstic acid negative staining solution (2%) onto the copper grid and let it stand for 3 min.

[0055] (5) Use the edge of the filter paper strip to absorb the excess phosphotungstic acid negative staining solution and dry it naturally until there is no liquid on the surface of the copper mesh.

[0056] (6) Observation was performed using a transmission electron microscope (Hitachi HT7800).

[0057] Result analysis: Naked colored microspheres from Figure 2 As can be seen in Figure A, the size is uniform, the particle size is 300nm, there is a brighter aperture on the surface of the colored microspheres, the edges are clear, and the whole is gray; Figure 2 Figure B shows that when the colored microspheres are coupled to the osteocalcin antibody K131c1 to prepare a probe, the antibody is coupled to the surface of the microspheres to increase the density of the microsphere particles, and the microspheres turn black as a whole. Due to the relatively low electron density of the protein antibody molecules, in contrast to the high electron density of the colored microsphere particles, there is a fuzzy and darker protein halo around the microspheres.

[0058] Example 4

[0059] Zeta potential analyzer measurement steps

[0060] The Omni (Brookhaven Instruments, USA) multi-angle particle size and high-sensitivity Zeta potential analyzer was used for determination. Prepare the test sample: Take 5 μL of the colored microspheres, dilute to 100 μL with pH 7.2 phosphate (PBS) buffer, and dilute to 5 mL with pH 7.2 PBS buffer. In addition, take 20 μL of the colored microsphere probe solution and dilute to 5 mL with PBS.

[0061] Put the colored microsphere dilution solution and the colored microsphere probe dilution solution into the cuvette, wipe the surface of the cuvette with paper, align the light-transmitting surface of the cuvette with the measurement area, set the relevant parameters, including the temperature setting of 25°C, the number of cycles of a single measurement is 20, the number of measurements is 3, and the single measurement interval is 0s, and then perform the measurement. The results are as follows Figure 3 As shown, the zeta potential of the bare colored microspheres is -22.89±2.25 mV, and the zeta potential of the colored microsphere probe is 51.73±3.79 mV, indicating that after the osteocalcin antibody K131c1 is coupled to the colored microspheres, the negative value of the potential changes significantly, proving that the osteocalcin antibody is successfully coupled to the surface of the colored microspheres.

[0062] Example 5

[0063] Osteocalcin Color Microsphere Test Strip Specificity Test

[0064] 1 μg / mL osteocalcin, Mycoplasma pneumoniae P1 adhesion protein, C-reactive protein (CRP), N-terminal pro-brain natriuretic peptide (NT-proBNP) and serum amyloid A (SAA) were prepared in pH 7.4BB buffer, and pH 7.4BB buffer was used as a negative control. According to the specific steps of the detection method of the colored microspheres for detecting osteocalcin test strips described above, the sample solution was replaced, mixed for 5 minutes, and allowed to stand for 2 minutes. The test strip was inserted into the enzyme-labeled well, and after chromatography for 12 minutes, the naked eye detection and mobile phone photography were used to record the experimental results.

[0065] The specific results of osteocalcin colored microsphere test strips are shown in Figure 4 , the serial numbers in the figure indicate: 1 is the positive control (containing osteocalcin); 2 is the negative control (without osteocalcin); 3 is the Mycoplasma pneumoniae P1 protein; 4 is CRP; 5 is NT-proBNP; 6 is SAA.

[0066] The results showed that test strip 1, as a positive control, had a band on the T line, while test strips 3-6 were consistent with test strip 2 (negative control) and only had a band on the C line. The results showed that the osteocalcin color microsphere test strip detection method had good specificity and had no cross-reaction with other proteins.

[0067] Comparative Example 1

[0068] Comparison of osteocalcin detection using the colored microspheres of the present invention and osteocalcin detection using the colloidal gold test strips of the prior art.

[0069] Preparation of colloidal gold test strips

[0070] (1) Preparation of immunocolloidal gold

[0071] Adjust pH: Take 1 mL of colloidal gold with a particle size of about 20 nm, add 5 μL of 0.2 MK 2 CO 3 , shake for 1 min.

[0072] Antibody: Add 12 μg of osteocalcin antibody (K131c1), shake at 500 g / min for 30 min.

[0073] Blocking: Add 100 μL of 10.0% bovine serum albumin (BSA), shake for 30 min, then centrifuge at 13000 g / min for 10 min, and discard the supernatant.

[0074] Add 500 μL of 1% BSA pH 7.4 BB buffer to the above precipitate, shake manually for 30 seconds, centrifuge at 13000 g / min for 10 minutes, and discard the supernatant.

[0075] Add 100 μL of 1% BSA pH 7.4 BB buffer to the above precipitate and shake for 30 seconds. The immunocolloidal gold preparation is complete and stored at 4°C for future use.

[0076] (2) Steps of colloidal gold immunochromatographic test strips

[0077] Assemble the sample pad, NC membrane, bottom plate and absorbent paper into a test strip, cut it into a width of about 4mm with a strip cutter, set the T line at the end of the NC membrane near the sample pad, and set the C line at the absorbent paper end of the NC membrane. The T line is dotted with K135c2, and the C line is dotted with goat anti-mouse IgG, with a dotted volume of 1μL; place it in an incubator at 37℃ for 1h. The concentration of the T line is 0.5mg / mL, and the concentration of the C line is 1mg / mL.

[0078] Take 5 μL of immune colloidal gold, 25 μL of osteocalcin sample solution with different concentrations (10000 ng / mL, 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156 ng / mL and 0 ng / mL) and 50 μL of 1% Tween 20 pH 7.4 BB buffer, add them into the microwells of the ELISA plate in sequence, mix for 5 minutes, and let it stand for 2 minutes. Then insert the prepared test strip into the ELISA well, and after chromatography for 12 minutes, take out the test strip and place it on the laboratory table to observe the results. Figure 5As shown in the figure, test strips 1 to 8 are different concentrations of osteocalcin: 1, 10000ng / mL 2, 5000ng / mL 3, 2500ng / mL 4, 1250ng / mL 5, 625ng / mL 6, 312.5ng / mL 7, 156ng / mL 8, 0ng / mL.

[0079] The test strip of Example 2 was used, the osteocalcin antibody K135c2 was used as the detection line (T line) to coat the original, and the goat anti-mouse IgG was used as the quality control line (C line) to coat the original and spot the sample on the NC membrane, and 1 μL of the sample was spotted with a pipette; and the sample was placed in an incubator at 37° C. for 1 hour. The coating concentration of the T line was 0.5 mg / mL, and the coating concentration of the C line was 1 mg / mL.

[0080] 25 μL of osteocalcin samples with different concentrations (osteocalcin concentration: 2-fold gradient dilution from 625 ng / mL to 4.9 ng / mL, with 0 ng / mL as control), 25 μL of colored microsphere probes and 15 μL of 1% Tween 20CBS solution were placed in the microwells of the ELISA plate, mixed for 5 minutes, left to stand for 2 minutes, and chromatographed for 12 minutes. The experimental results were recorded by naked eye detection and mobile phone photography. Figure 6 As shown, test strips 1 to 9 are different concentrations of osteocalcin: 1, 625 ng / mL 2, 312.5 ng / mL 3, 156 ng / mL 4, 78 ng / mL 5, 39 ng / mL 6, 19.5 ng / mL 7, 9.8 ng / mL 8, 4.9 ng / mL 9, 0 ng / mL.

[0081] Compared with the minimum detection limit of 156ng of the colloidal gold osteocalcin immunochromatographic test strip, the minimum detection limit of the colored microsphere osteocalcin immunochromatographic test strip is 4.9ng, and the sensitivity is increased by nearly 31 times.

[0082] Comparative Example 2

[0083] During the coupling process of the three colored microspheres, due to the different isoelectric points of different antibodies and the different charges carried by antibodies in different buffers, in order to screen the best buffer for coupling of colored microspheres and antibodies, three kinds of colored microsphere probes were prepared using pH 6.0 MES buffer, pH 7.2 PBS buffer and pH 7.4 BB buffer, and comparative experiments were conducted on the coupling effect and detection effect of the probes.

[0084] Three colored microspheres were prepared and treated according to the method of Example 1. After washing in step C, 50 μL of pH 6.0 MES buffer (labeled as microsphere probe 1), PBS buffer (labeled as microsphere probe 2) and pH 7.4 BB buffer (microsphere probe 3) were added to resuspend the microspheres, and then the subsequent steps were carried out according to the method of Example 1.

[0085] Results Figure 7 , see the effect ranking Figure 7 A (Serial numbers in the figure: 1. Boric acid colored microsphere probe 3; 2. Colored microsphere probe 2; 3. Colored microsphere probe 1). It can be seen from the figure that colored microsphere probe 3 has the best coupling effect, and there is basically no granular material sticking to the wall of the EP tube, and the supernatant is clear. However, colored microsphere probe 2 and colored microsphere 1 have different degrees of colored microsphere sticking to the wall, and the colored microsphere 1 sticking to the wall is more obvious, indicating that the granular colored microspheres are agglomerated. Therefore, in terms of effect, tube 1 (colored microsphere probe 3)> tube 2 (colored microsphere probe 2)> tube 3 (colored microsphere probe 1). This phenomenon may be caused by the fact that the acidic buffer solution has a certain effect on the charge on the surface of the microspheres, and the colored microspheres are aggregated and precipitated.

[0086] In order to further verify the effect of preparing the probe, we assembled the test strips with the sample pad, bottom plate, NC membrane and absorbent paper according to the steps of the previous test strips. The T line is near the end of the sample pad on the NC membrane, and the C line is at the end of the absorbent paper on the NC membrane. K135c2 is dotted on the T line, and goat anti-mouse IgG is dotted on the C line. The sample volume is 1μL; it is placed in an incubator at 37℃ for 1h. The concentration of the T line is 0.5mg / mL, and the concentration of the C line is 1mg / mL. The test strips are dried for 1h. Then, according to the following reagent configuration, they are placed in 6 enzyme-labeled wells.

[0087] (1) Well 1: 25 μL 10-fold diluted microsphere probe 1 + 25 μL sample (200 ng / mL osteocalcin) + 15 μL 1% Tween CBS buffer.

[0088] (2) Well 2: 25 μL of 10-fold diluted microsphere probe 1 + 25 μL of BBB buffer (without osteocalcin) + 15 μL of 1% Tween CBS buffer.

[0089] (3) Well 3: 25 μL 10-fold diluted microsphere probe 2 + 25 μL sample (200 ng / mL osteocalcin) + 15 μL 1% Tween CBS buffer.

[0090] (4) Well 4: 25 μL of 10-fold diluted microsphere probe 2 + 25 μL of BBB buffer (without osteocalcin) + 15 μL of 1% Tween CBS buffer.

[0091] (5) Well 5: 25 μL 10-fold diluted microsphere probe 3 + 25 μL sample (200 ng / mL osteocalcin) + 15 μL 1% Tween CBS buffer.

[0092] (6) Well 6: 25 μL of 10-fold diluted microsphere probe 3 + 25 μL of BBB buffer (without osteocalcin) + 15 μL of 1% Tween CBS buffer.

[0093] Shake for 5 minutes, let stand for 2 minutes, then insert the test strip into the above enzyme-labeled well and chromatograph for 12 minutes. Figure 7 B (Serial numbers in the figure: 1. Colored microsphere probe 1, positive sample; 2. Colored microsphere probe 1, negative sample; 3. Colored microsphere probe 2, positive sample; 4. Colored microsphere probe 2, negative sample; 5. Colored microsphere probe 3, positive sample; 6. Colored microsphere probe 3, negative sample). Microsphere probe 1 has the worst effect, and the T line can be vaguely seen. Microsphere probe 2 is better, and the naked eye effect of the T line is better than that of microsphere probe 1, but worse than that of microsphere probe 3. Microsphere probe 3 has the best effect, and the T line and C line are the most obvious and clearly visible. The results show that BB buffer is the best coupling buffer for colored microsphere probes.

[0094] Experimental Example 1

[0095] During the activation process, the concentrations of EDC and NHS are related to the carboxyl groups on the surface of the microspheres. In general, when the concentrations of EDC and NHS are high, the carboxyl groups on the surface of the microspheres are easily over-activated. Based on this, we used high concentrations of EDC (20 mg / mL) and NHS (20 mg / mL) according to the published literature (Lin J, Li S, Ye B, et al. A time-resolved fluorescence microsphere-lateral flow immunochromatographic strip for quantitative detection of Pregnanediol-3-glucuronide in urine samples [J]. Frontiers in Bioengineering and Biotechnology, 2023, 11: 1308725.), and the other group used low concentrations of EDC (1 mg / mL) and NHS (1 mg / mL) for simultaneous activation. The specific usage is shown in Table 1. The results showed that during the coupling process, neither of the two showed the phenomenon of colored microsphere aggregation and precipitation and EP tube wall hanging. The intermediate coupling solution was uniform, and there was no obvious difference between the two. Therefore, the present invention adopts EDC (1 mg / mL) and NHS (1 mg / mL) with a lower concentration for coupling, which saves the amount of EDC solution and NHS and reduces the cost.

[0096] Table 1 Comparison of concentrations of EDC and NHS used in microsphere coupling

[0097] Microsphere amount EDC NHS Prior art 5μL 20mg / mL,5μL 20mg / mL,5μL The present invention 5μL 1mg / mL,2.4μL 1mg / mL,2.8μL

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a colored microsphere probe, characterized in that: The steps include: (1) washing the colored microspheres and centrifuging to obtain a precipitate; (2) Activating the microspheres in the precipitate; (3) After washing again, resuspend the microspheres in borate buffer; (4) The resuspended microspheres are coupled with osteocalcin antibodies, and then blocked and stored.

2. The preparation method according to claim 1, characterized in that: The cleaning in step (1) is performed using a boric acid buffer solution at pH 7.4-pH 8.0, and the volume ratio of the colored microspheres to the boric acid buffer solution is 1-10:

100.

3. The preparation method according to claim 2, characterized in that: The activation in step (2) is to resuspend the precipitated microspheres in a boric acid buffer solution of pH 7.4-pH 8.0, add EDC solution and NHS solution, and continue for 25-35 minutes under the condition of a metal bath at 35°C-39°C.

4. The preparation method according to claim 3, characterized in that: During the activation, the concentration of the EDC solution is 0.8-1.2 mg / ml, the concentration of the NHS solution is 0.8-1.2 mg / ml, and the volume ratio of the boric acid buffer solution, the EDC solution and the NHS solution is 90-110:2.2-2.6:2.6-3.

5. The preparation method according to claim 4, characterized in that: The second washing in step (3) is performed using boric acid buffer at pH 7.4-pH 8.0, wherein the added volume of the boric acid buffer is 4-6 times the volume of the boric acid buffer added during activation. After the washing, the mixture is centrifuged again and resuspended with boric acid buffer at pH 7.4-pH 8.0, wherein the volume of the resuspending buffer is 1 / 9-1 / 10 of the volume of the boric acid buffer during the above washing.

6. The preparation method according to claim 5, characterized in that: The antibody in step (4) is K131c1, the concentration of K131c1 is 3.5-4.5 mg / mL, the volume ratio of K131c1 added to the resuspension buffer is 1-3:50-60, the coupling conditions are in a metal bath at 35°C-39°C for 55-65 min, the blocking is followed by centrifugation, and then the preservation solution is added, the preservation solution is 1-3% BSA pH7.4-pH8.0 boric acid buffer.

7. A colored microsphere probe obtained by the preparation method according to any one of claims 1 to 6.

8. A test strip prepared using the colored microsphere probe according to claim 7.

9. The test strip according to claim 8, characterized in that: The test strip comprises a nitrocellulose membrane, a sample pad and absorbent paper which are attached to a bottom plate, and the detection line of the nitrocellulose membrane is coupled with an osteocalcin antibody K135c2.

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